Recombinant Mouse Granzyme D Protein, CF

• Reactivity: Mu
• Applications: Enzyme Activity
• Format: Carrier-Free

Recombinant Mouse Granzyme D Protein, CF Summary

• Details of Functionality: Measured by its ability to cleave a peptide substrate, Succinyl-Phe-Leu-Phe-ThioBenzyl ester (Suc-FLF-SBzl), in the presence of 5,5’-Dithio-bis (2-nitrobenzoic acid) (DTNB). Edwards, K.M. et al. (1999) J. Biol. Chem. 274:30468. The specific activity is >50 pmol/min/µg, as measured under the described conditions.
• Source: Mouse myeloma cell line, NS0-derived mouse Granzyme D protein
  Ile21-Leu252 with a C-terminal 10-His tag
• Accession #: AAA37736
• N-terminal Sequence: Ile21 & His25
• Protein/Peptide Type: Recombinant Enzymes
• Gene: Gzmd
• Purity: >95%, by SDS-PAGE under reducing conditions and visualized by silver stain
• Endotoxin Note: <1.0 EU per 1 μg of the protein by the LAL method.

Applications/Dilutions

• Dilutions:
  • Enzyme Activity
• Theoretical MW: 27 kDa.
  Disclaimer note: The observed molecular weight of the protein may vary from the listed predicted molecular weight due to post translational modifications, post translation cleavages, relative charges, and other experimental factors.
• SDS-PAGE: 42 kDa, reducing conditions

Packaging, Storage & Formulations

• Storage: Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
  • 6 months from date of receipt, -20 to -70 °C as supplied.
  • 3 months, -20 to -70 °C under sterile conditions after reconstitution.
• Buffer: Lyophilized from a 0.2 μm filtered solution in HEPES and NaCl.
• Purity: >95%, by SDS-PAGE under reducing conditions and visualized by silver stain
• Reconstitution Instructions: Reconstitute at 100 μg/mL in sterile 25 mM HEPES and 200 mM NaCl, pH 7.5.
• Assay Procedure:
  • Assay Buffer: 0.1 M Tris, pH 9.0
  • Recombinant Mouse Granzyme D (rmGranzyme D) (Catalog # 1365-SE)
  • Substrate: SUC-Phe-Leu-Phe-SBZL (Bachem, Catalog # M-1740), 10 mM in DMSO
  • 5,5’-dithio-bis (2-nitrobenzoic acid) (DTNB) (Sigma, Catalog # D-8130), 10 mM in DMSO
  • 96 well Clear Plate (Costar, Catalog # 92592)
  • Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
  1. Preheat plate reader and Assay Buffer at 37 °C.
  2. Dilute rmGranzyme D to 2 ng/µL in preheated Assay Buffer.
  3. Combine equal volumes of Substrate and DTNB for final concentrations of 5 mM of each.
  4. Dilute the Substrate/DTNB mixture to 200 µM in preheated Assay Buffer.
  5. Load 50 µL of the diluted rmGranzyme D into a 96 well clear plate. Include a Substrate Blank containing 50 µL of Assay Buffer.
  6. Start the reaction by adding 50 µL of the diluted Substrate/DTNB mixture to wells.
  7. Read at a wavelength of 405 nm (bottom read) in kinetic mode for 5 minutes.
  8. Calculate specific activity:

  Specific Activity (pmol/min/µg) = (Adjusted Vmax* (OD/min) x well volume (L) x 10^12 pmol/mol) / (ext. coeff** (M^-1cm^-1) x path corr.*** (cm) x amount of enzyme (µg))

  
  *Adjusted for Substrate Blank
  **Using the extinction coefficient 13260 M^-1cm^-1
  ***Using the path correction 0.32 cm
  Note: the output of many spectrophotometers is in mOD. Per Well:
  • rmGranzyme D: 0.1 µg
  • Substrate: 100 µM
  • DTNB: 100 µM

Notes

This product is produced by and ships from R&D Systems, Inc., a Bio-Techne brand.

Alternate Names for Recombinant Mouse Granzyme D Protein, CF

• CCP5
• Ctla5
• Ctla-5
• Granzyme D
• GrzD

Background

Granzyme D is a member of the granzyme family of the serine proteases found specifically in the cytotoxic granules of cytotoxic T lymphocytes (CTL) and natural killer (NK) cells (1-3). Granzyme D’s functions are largely unknown. However, its selective expression in primary and functional hematopoietic stromal lines correlates with capacity of supporting growth of a pre-T lymphoma clone (4). Together with Granzymes E, F and G, it is regulated through pregnancy and by IL-2 and IL-15 in granulated metrial gland cells (5). The human or rat counterpart of mouse Granzyme D has not been found. Like other granzymes, mouse Granzyme D is not secreted as a zymogen but stored as a fully processed and activated enzyme in the cytoplasmic granules of CTL (1). It is synthesized as a 252 amino acid precursor with an 18 amino acid signal peptide and a 2 amino acid propeptide (1). The mature protein (residues 21-252) is expressed and purified.

1. Jenne, D.E. et al. (1988) Proc. Natl. Acad. Sci. USA 85:4814.
2. Kam, C.-M. et al. (2000) Biochim. Biophys. Acta 1477:307.
3. Smyth, M.J. et al. (1996) J. Leukoc. Biol. 60:555.
4. Yokoi, A. et al. (1999) Biochem. Biophys. Res. Comm. 264:768.
5. Allen, M.P. and M. Nilsen-Hamilton (1998) J. Immunol. 161:2772.

 

Bioinformatics

• Gene Symbol: Gzmd
• Uniprot:
  • AAA37736()